Successful targeting in situ of an oncogenic nuclear antigen by hapten induced tumor associated autoantibodies (iTAA)

The abscopal is a hypothesis for treating of non-irradiated tumors after localized radiation therapy. It is associated with the products of tumor-associated gene as autoantibodies (aTAAs) in reaction to the tumor-associated antigens (TAAs), with increasing of anti-MAGEA3 and an relationship between the abscopal effect and immune response. The hapten enhanced local chemotherapy (HELC) was studied to kills tumor and release tumor TAAs, then hapten modify the TAAs to neu-TAAs, to produce tumor autologous antibodies, called induced tumor-associated autoantibodies (iTAAs) that is different from natural TAAs. Since the iTAAs and complement (C) are associated with cancer therapy Immunofluorescence (IF) was applied to evaluate the expression of the iTAAs and C3, C5, C9. Traces resulted in a partial staining of the nucleus in C3’s perinuclear reaction. The iTTAs of Survivin, C-MYC, and IMP1 increased significantly in the tumor cells' intranuclear regions (P = 0.02, P = 0.00, P < 0.0001). Koc, zeta, RalA, and p53 had a similar trend in the perinuclear regions (P < 0.0001, P = 0.004, P < 0.0001, P = 0.003). Therefore, we can propose that tumor antigens inside the cancer cells’ nuclei are targeted by the iTAAs since the iTAAs binding levels are higher after HELC. The iTAA tagging oncogenic nuclear antigens may play a distinctive role in regulating tumor cell growth.

are produced and how this circulation may connect with their relationship to curative treatments or abscopal. However, the iTAAs share a connection with therapeutic cancer treatments and may play into the distal abscopal effect of tumors 9,15,16 . Hence, a preliminary investigation of the iTAAs in cancer patients revealed an increase in autologous antibodies of TAA: HCC1, RalA, zeta, and p16 17 . These results point toward the rationale that the increase may be related to extend survival time with the abscopal effect 9,18 . So far, scientists do not know how to track iTAAs that result from distal tumor cells or if the iTAAs can enter tumor cells. If they are to enter the tumor cells, researchers do not know wheather the iTAAs' ability to enter the cells is associated to the complement response which opens the tumor membrane.
We specifically selected representatives of zeta, IMP1, Koc, Survivin, c-MYC, RalA, and p53 gene as marker for research, each gene may have different function in the tumorgenesis, among them, the p53 cancer suppressor gene was a very hot gene studied in the last century, we aimed to provide evidence to prove that HELC treatment induces tumor responses in cancer patients to produce iTAA. Furthermore, we aimed to decipher if the interdependence of tumor cell membranes is an adjuvant function of complement and if the iTAAs induced in the body travel to tumor cells. If so, we intended to determine which location. Finally, we sought to determine whether the iTAAs enter tumor cells at their primary tumor of primary stage or later during metastasis. This focus was set in an effort to advance our understanding of the relationship between iTAAs and abscopal effect.

Materials and methods
Clinical specimens. The patients received HELC treatment at Taimei Baofa Cancer Hospital. Each had a precise clinical diagnosis, met the indications for HELC chemotherapy, signed the informed consent form, and this experiment was approved by the hospital ethics committee Taimei Baofa Cancer Hospital (TMBF 0010, 2015) for therapy and participation in the study prior to either commencing and all method for experiments were performed in accordance with relevant guidelines and regulations 4,17 .
A total of seven patients with tumors were included in the study. The cases included three non-small cell lung cancer (NSCLC) patients, two esophageal squamous cell carcinoma (ESCC) patients, one cervical squamous cell carcinoma patient, and one left parotid gland malignancy patient. Each received an HELC treatment which consists of combination and off label use with adriamycin, cytarabine, hydralazine as hapten, final concentration is 1.0 mg + 0.8 mg + 1.0 mg + 7.2 mg/ml (Total dose 5 ml), the injection needle tip in the tumor was monitored by CT 4,9,10 . Biopsy samples were collected by biopsy needle from all of the patients one weeks before and one to two weeks after their HELC treatment, all sample is very small and limited to section more than 10 slides so that sometimes IF staining cannot performed for each sample by 7 tumor antigens.
To observe the abscopal effect, the biopsy sites were the core of untreated tumor or metastased lymph node after primary tumor HELC treatment for three NSCLC, one cervical squamous cell carcinoma, and two ESCC, one left parotid gland malignancy (Table 1). A few untreated tumor samples before HELC treatment is used for control. Once biopsied, the clinical specimens were immediately preserved in formalin, embedded in paraffin, and sectioned for IF staining while some patient's blood collected for measure the level of iTAA. Antibody detection analysis. An enzyme-linked immunosorbent assay (ELISA) was used to assess the signals of 7 purified recombinant proteins in phosphate-buffered saline (PBS). The final concentrations ranged from 0.125 ug/ml to 1.0 ug/ml. The proteins were then coated in a 96-well microliter plate (100ul/well) overnight at 4 °C and incubated in a 1:200 diluted serum in antigen-coated wells (100ul/well) for 90 min at room temperature (RT). Each well's optical density (OD) value was immediately read at 405 nm on the Varioskan LUX Multimode Microplate Reader to reduce the plates' variation 19,20 . IF: imaging and analysis. After staining was complete, each section was photographed at Shandong University with a multispectral panoramic tissue scanning microscope (TissueFAXS Spectra). An individual blinded to this study and had no conflict of interest performed this photography and then conducted the fluorescence imaging and data analysis. The Tissue FAXS Viewer software was used for processing, and the images were exported after adjusting the lower and upper values of the image range before and after treatment to be consistent 21 .
Statistical analysis. The expression differences of complement factors, as well as the factors of iTAAs were analyzed in the tissues before and after treatment using GraphPad Prism v8.0.2.263.21 A paired t-test was used to determine the percentage of positive cells and immunofluorescence intensity (MFI) and P < 0.05 indicated a statistically significant difference.

Positive complement cells and iTAAs' mean immunofluorescence intensity (MFI) rates.
Before and after treatment, analysis of the positive staining of the complement, the 7 of TAA-bearing fluorescein and mean MFIs were executed for each of iTAAs in all tumor sections to show where each of complement C and iTAAs was being. The percentage of the positive complement was higher after than before for C3 ( www.nature.com/scientificreports/

Discussion
The aTAAs are often produced in the human body but maintain a low-level presence 8,11 . The number of aTAAs may surge if a virus mutates the TAAs 11 . The mutation can occur when viruses or mutated proteins from oncogenes or genes other than those originating from the human body cause tumors 8,11 . Otherwise, aTAAs can be generated through the injection of hapten modified with TAA. This modified TAA possesses a slight change on its epitope so it is recognized by the immune system as a neoantigen, and thus incites a humoral response 4,11,20 .  www.nature.com/scientificreports/ Moreover, an antibody's amplification response to the presence of an antigen means that even a small quantity of antigens in the early stage of tumorigenesis can trigger a relatively large immune response 4,22 . Therefore, aTAAs are feasible early diagnostic markers. Despite using aTAAs as biomarkers for clinical diagnosis, the iTAAs should be studied in detail for application to cancer treatments (Fig. 24).  www.nature.com/scientificreports/ We established that HELC therapy induces an immune response from tumors with DC, CD4 and CD8 positive tumor tissue after treatment 4,9,10 . We found a dendritic cell (DC) using an electric microscope and the DC11b/c expression was increased in our tumor mice model 4,9,10 . DC and T cells of whole immunity systems www.nature.com/scientificreports/ were activated following HELC. Therefore, we believe that B cells of immunity systems must be activated as well at same times, so the iTAAs are produced following immunity reaction induced by neu TAA in the patient. Follow the iTAAs' activated production, they analyze, target, and then bind to tumor cells. Thus, this study confirms that the iTAAs are induced and excited in the sera after HELC treatment ( Table 2, Fig. 25), and iTAAs can circulate from blood to bind the tumor at the cellular level (Figs. 6, 7, 8, 9, 10, 11) since the iTAA has a high specificity to  www.nature.com/scientificreports/ Since the aTAAs were not found an increase and high specificity binding of in the patients' tumor cells before treatment of HELC, further confirmation emerges supporting the assertion that the iTAAs increase in presence with a higher specificity after the treatment courses and target critical locations. The IMP1, c-MYC, and Survivin increased significantly in the intranuclear tumor cell locations. In contrast, Zeta, Koc, RalA, and p53 were  Table 4). The seven iTAAs we studied are representative of countless tumor gene products induced by hapten (Tables 3, 4). More gene products may be able to induce more significant quantities  www.nature.com/scientificreports/ gene indicator of malignancy; Survivin is essential for cell division and can inhibit cell death; c-myc is of great importance in controlling cell growth and vitality; RALA is highly homologous small G proteins belonging to the RAS superfamily; p53 is the most frequently mutated gene across all cancer types, its functions has evolved since its discovery four decades ago, current knowledge of p53 functions derived through the major classes of anti-p53 antibodies, which could be a paradigm for understanding other molecular events in health and disease [23][24][25][26][27][28][29] . However, the method used is an indirect of assess the signals of iTAAs for 7 maker genes as representatives, so that limitations of our study approach needs to improve in the future study. A hypothesis is that iTAAs bind the TAAs in nuclear of tumor cells may feedback to regulate the expression of different genes depending on what genes function while the iTAAs bind the TAA in blood could do nothing. The iTAAs, the study's results establish that, once induced, iTAAs circulate in the blood to search for the primary tumor or metastasis site to bind to the nuclei of those tumor cells. We can propose that this process is a result of an abscopal effect.
Investigation of how these iTAA-bound tumor cells survive, die faster, live better, or live differently is required. Future studies might involve collecting circulating tumor cells (CTCs) from cancer patients' post-HELC treatment and then culturing the CTCs for analysis with IF using TAA-bearing fluorescein, followed by analysis of how the CTCs live. Through sequencing and proteomics, evaluation of the expression of TAAs' different proteins, including their DNA and RNA, is critical. Finally, further detailed studies, with and without hapten-enhanced intratumoral injections in patients are required to develop a comprehensive understanding of the full diagnostic and therapeutic potential of the iTAAs. Table 4. Comparison of intranuclear and perinuclear positive staining and targeting ratios before and after each factor treatment. www.nature.com/scientificreports/       www.nature.com/scientificreports/

Data availability
The data that support the findings of this study are available from [third party name] but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with writing permission of [third party name] to Baofa Yu.  Before treatment After treatment Figure 24. Trend analysis of expression autoantibodies aTAA before treatment and iTAA after treatment, complement levels before and after treatment. www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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